Co-culture of Human Embryonic Stem Cell derived Osteoprogenitors and Endothelial Cells

Objectives: Human Embryonic Stem Cells (hESC) are potential unlimted source of Osteoprogenitors (OP) and Endothelial Cells (EC). The differentiation efficiency, homogeneity and use of animal products are challenges that need to be addressed.
The objective was to develop efficient protocols for derivation of hESC-OP and hESC-EC with minimal use of xenogenic products and to see if hESC-OP can be used as supporting cells for EC differentiation.
Methods: Under feeder-free and serum-free conditions, hESC were differentiated to hESC-OP through stages encompassing embryoid bodies and embryoid body outgrowth. The differentiation of hESC to hESC-OP was confirmed at transcript and protein levels, and differentiation towards osteogenic lineage under osteogenic conditions. Similarly, hESC were differentiated towards hESC-EC under feeder-free and serum-free conditions, using sequential modulation of Wnt, FGF, BMP and VEGF signaling pathways. The characteristics of hESC-EC were confirmed at transcript and protein levels and tube formation assays. The in-vitro functionality of these hESC-OP and hESC-EC was investigated using 2-dimensional (2d) and 3-dimensional (3d) co-culture systems. To enable visualization of hESC-OP and hESC-EC under co-culture conditions, hESC-OP were labelled with red fluorescent protein (DsRed2) and hESC-EC with green fluorescent protein.
Results: The protocol resulted in greater efficiency in the generation of EC. 2d co-culture of hESC-OP and hESC-EC demonstrated the cell-cell interaction of both cell types resulting in reorganization of hESC-EC into anastomosing network of endothelial cords. Similarly, 3d co-culture of fluorescently labelled hESC-OP and hESC-EC within fibrin-based matrix resulted in formation of an anastomosing network of hESC-EC derived vascular channels supported by hESC-OP.
Conclusions: This is the first instance of successfully using hESC-OP as supporting cells for EC differentiation. From a future prospective, we believe that the 3d co-culture of hESC-OP and hESC-EC within appropriate scaffold will pave the way for generation of in vitro prevascularized bone constructs.